NFATC2IP Antibody

What is NFATC2IP and what cellular functions does it regulate?

NFATC2IP (also known as NIP45) is a 419 amino acid protein containing one ubiquitin-like domain. This protein plays critical roles in:

• Regulating NFAT-driven transcription of specific cytokine genes (IL3, IL4, IL5, and IL13, but not IL2) in T-helper 2 (Th2) cells

• Recruiting PRMT1 to the IL4 promoter, which enhances histone H4 'Arg-3'-methylation and facilitates histone acetylation, promoting robust cytokine expression

• Down-regulating formation of poly-SUMO chains by UBE2I/UBC9

• Promoting genome integrity and cellular survival in response to SUMOylation inhibition

The protein contains important structural features including SLD1 and SLD2 domains, with the SLD2 domain being critical for its function in promoting survival during SUMOylation inhibition .

What applications are NFATC2IP antibodies validated for?

NFATC2IP antibodies have been validated for multiple research applications:

What are the recommended dilutions for NFATC2IP antibodies in different applications?

The following dilutions are recommended based on validated antibodies:

Note: It is strongly recommended that researchers titrate each antibody in their specific testing system to achieve optimal results .

How should I design proper controls when using NFATC2IP antibodies?

Effective controls are essential for research using NFATC2IP antibodies:

• Positive controls: Use cell lines where NFATC2IP expression has been confirmed, such as:

  • Jurkat cells, K-562 cells, and Raji cells for Western blot

  • HeLa cells for immunofluorescence

• Negative controls: Consider using:

  • NFATC2IP knockout cells (as demonstrated in research using clonal knockout RPE1-hTERT TP53-/- Cas9 cells with biallelic one-nucleotide deletion c.493delC)

  • Primary antibody omission controls

  • Isotype controls (Rabbit IgG for polyclonal antibodies)

• Validation controls:

  • Overexpression systems using NFATC2IP-transfected cells versus non-transfected lysates

  • Peptide competition assays to confirm specificity

What are the critical parameters for optimizing Western blot protocols with NFATC2IP antibodies?

Optimizing Western blot protocols for NFATC2IP detection requires attention to several key parameters:

• Molecular weight expectations:

  • Calculated molecular weight: 15 kDa (138 aa)

  • Observed molecular weight: 50-65 kDa

  • This discrepancy suggests post-translational modifications or complex formation

• Sample preparation:

  • Use fresh samples or properly stored frozen lysates

  • Include protease inhibitors to prevent degradation

  • Consider phosphatase inhibitors if studying phosphorylation status

• Optimization recommendations:

  • Start with 1:500 dilution and adjust based on signal strength

  • Validated in Jurkat cells, K-562 cells, and Raji cells

  • Use 4-20% gradient gels for optimal separation

  • Extended transfer time may be required for complete protein transfer

• Detection considerations:

  • Enhanced chemiluminescence (ECL) is suitable for most applications

  • Fluorescent secondary antibodies may provide better quantification

What are the best practices for immunofluorescence detection of NFATC2IP?

For optimal immunofluorescence results when studying NFATC2IP:

• Cell preparation:

  • HeLa cells have been validated for NFATC2IP detection by IF

  • Consider using 4% paraformaldehyde fixation followed by 0.1% Triton X-100 permeabilization

• Antibody dilutions:

  • Start with 1:200-1:800 dilution range

  • Titrate to determine optimal signal-to-noise ratio

• Controls:

  • Include secondary antibody-only controls to assess background

  • Consider co-staining with markers of known subcellular compartments to confirm localization

• Visualization:

  • NFATC2IP may show both nuclear and cytoplasmic localization depending on cellular context

  • Confocal microscopy is recommended for precise subcellular localization studies

How can I use NFATC2IP antibodies to study its role in T-helper cell cytokine regulation?

NFATC2IP plays a crucial role in regulating cytokine expression in Th2 cells. The following experimental approach is recommended:

• Cell model selection:

  • Primary CD4+ T cells differentiated under Th2 conditions

  • Jurkat cells for initial optimization

• Experimental design:

  • Compare NFATC2IP localization before and after T cell activation using IF

  • Perform ChIP assays using NFATC2IP antibodies to assess binding at IL3, IL4, IL5, and IL13 promoters

  • Conduct co-IP experiments to study interactions with NFAT proteins and PRMT1

• Functional validation:

  • Use siRNA knockdown or CRISPR knockout of NFATC2IP

  • Measure cytokine production by ELISA or intracellular cytokine staining

  • Assess histone H4 'Arg-3'-methylation at the IL4 locus using ChIP

• Data interpretation:

  • Correlate NFATC2IP binding with cytokine gene expression

  • Analyze the impact of NFATC2IP depletion on NFAT-dependent transcription

How can I investigate NFATC2IP's role in SUMOylation pathways?

Recent research has identified NFATC2IP as important in SUMOylation pathways . To investigate this function:

• Experimental model:

  • Consider using RPE1-hTERT cells as used in published research

  • Generate NFATC2IP knockout cells using CRISPR/Cas9 editing targeting frameshift mutations (e.g., c.493delC)

• Functional assessment:

  • Treat cells with SUMOylation inhibitors (e.g., TAK-981) and assess survival

  • Measure cellular sensitivity using clonogenic survival assays

  • Assess micronucleation as a marker of genomic instability

• Structure-function analysis:

  • Express wild-type NFATC2IP or domain mutants:

    • Complete deletion of SLD2 domain (ΔSLD2)

    • Point mutation D394R (disrupts interaction with UBC9)

    • SLD1 domain deletion (ΔSLD1)

  • Assess rescue of phenotypes in knockout cells

• Protein interaction studies:

  • Perform co-IP with UBC9/UBE2I to study direct interactions

  • Assess global SUMOylation patterns in the presence/absence of NFATC2IP

What methodological considerations are important when studying the different domains of NFATC2IP?

NFATC2IP contains important functional domains including SLD1 and SLD2. When investigating these domains:

• Antibody selection:

  • Choose antibodies that recognize different regions of NFATC2IP

  • For N-terminal studies: consider antibodies targeting AA 1-138

  • For C-terminal/SLD2 studies: select antibodies targeting regions containing the SLD2 domain

• Domain-specific constructs:

  • Generate expression constructs for domain-specific studies:

    • Full-length NFATC2IP (control)

    • ΔSLD2 (deletion of SLD2 domain)

    • D394R point mutant (disrupts UBC9 interaction)

    • ΔSLD1 (deletion of SLD1 domain)

• Functional assays:

  • TAK-981 sensitivity assays show that SLD2 is critical for survival during SUMOylation inhibition

  • Micronucleation assays reveal that both SLD1 and SLD2 contribute to genomic stability, with SLD2 playing a more critical role

• Data interpretation challenges:

  • Different domains may contribute to different functions

  • Consider both structural and protein-interaction roles of each domain

Why might I observe different molecular weights for NFATC2IP in Western blot experiments?

NFATC2IP shows a discrepancy between calculated and observed molecular weights:

• Expected vs. observed weights:

  • Calculated molecular weight: 15 kDa (138 aa)

  • Observed molecular weight: 50-65 kDa in Western blots

• Potential explanations:

  • Post-translational modifications (SUMOylation, phosphorylation)

  • Alternative splicing generating larger isoforms

  • Dimerization or complex formation that resists denaturation

  • The full protein length is actually 419 amino acids, which may explain the higher observed weight

• Verification approaches:

  • Use denaturing conditions with reducing agents

  • Compare with overexpressed tagged NFATC2IP

  • Consider 2D gel electrophoresis to separate based on both size and charge

How can I optimize antibody performance for detecting endogenous NFATC2IP in different cell types?

Different cell types may require customized approaches for optimal NFATC2IP detection:

• Cell type considerations:

  • Validated cell lines include Jurkat, K-562, Raji (WB) and HeLa (IF)

  • Expression levels may vary significantly between cell types

  • T-helper cells show functional significance of NFATC2IP

• Sample preparation optimization:

  • For adherent cells: direct lysis in well vs. scraping and pelleting

  • For suspension cells: ensure adequate cell numbers (1-5 × 10^6 cells/mL)

  • Compare different lysis buffers (RIPA vs. NP-40)

• Signal enhancement strategies:

  • Increase antibody concentration for low-expressing cells

  • Use signal amplification systems for weak signals

  • Consider extended exposure times while monitoring background

• Background reduction:

  • Increase blocking time/concentration

  • Use antibody diluents with background reducers

  • Include additional washing steps

What approaches can resolve inconsistent results when using different NFATC2IP antibodies?

When different antibodies yield inconsistent results:

• Epitope considerations:

  • Different antibodies target different regions of NFATC2IP

  • Some antibodies target N-terminal regions (AA 1-138)

  • Others may recognize internal or C-terminal regions

• Validation approaches:

  • Use NFATC2IP knockout cells as negative controls

  • Perform peptide competition assays

  • Compare results with tagged overexpression constructs

• Application-specific optimization:

  • Some antibodies may work better for WB than IF

  • Adjust protocols based on each antibody's optimal application

  • Consider using multiple antibodies to confirm findings

• Reconciliation strategies:

  • When results differ, prioritize data from antibodies with stronger validation

  • Consider the possibility that different antibodies detect different isoforms or modified forms

How can NFATC2IP antibodies be used to study T-cell mediated inflammatory diseases?

Given NFATC2IP's role in regulating Th2 cytokines, it has potential implications for inflammatory and allergic conditions:

• Disease relevance:

  • NFATC2IP regulates IL4, IL5, and IL13, which are critical in allergic and asthmatic responses

  • It may represent a target for therapeutic intervention in Th2-driven conditions

• Experimental approaches:

  • Compare NFATC2IP expression and localization in normal vs. disease-state T cells

  • Assess correlation between NFATC2IP levels and cytokine production in patient samples

  • Study the effects of NFATC2IP modulation on disease phenotypes in animal models

• Technical considerations:

  • Use flow cytometry with NFATC2IP antibodies to analyze specific immune cell populations

  • Combine with cytokine staining to correlate NFATC2IP with functional outputs

  • Consider tissue immunostaining to assess NFATC2IP in affected tissues

What are the key considerations when investigating NFATC2IP's role in genomic stability pathways?

Recent findings link NFATC2IP to SUMOylation and genomic stability :

• Experimental models:

  • NFATC2IP knockout cells show hypersensitivity to SUMOylation inhibition

  • These cells demonstrate increased micronucleation, indicating genomic instability

• Research approaches:

  • Assess DNA damage markers in NFATC2IP-deficient cells

  • Investigate cell cycle progression and checkpoint activation

  • Evaluate chromosomal abnormalities using cytogenetic techniques

  • Study NFATC2IP localization during different cell cycle phases

• Mechanistic investigations:

  • Examine interactions between NFATC2IP and DNA repair proteins

  • Study the role of the SLD2 domain in promoting genomic stability

  • Investigate whether NFATC2IP directly affects SUMOylation of specific target proteins

How can I combine NFATC2IP antibodies with CRISPR-based approaches for functional studies?

Integrating CRISPR technology with antibody-based detection offers powerful research opportunities:

• Genetic manipulation approaches:

  • Generate NFATC2IP knockout cell lines using CRISPR/Cas9

    • Target frameshift mutations (e.g., c.493delC causing p.His165MetfsX15)

    • Create domain-specific mutations

  • Use CRISPR interference (CRISPRi) for transient repression

• Validation strategies:

  • Confirm knockout using NFATC2IP antibodies in Western blot

  • Verify loss of nuclear localization using immunofluorescence

  • Assess functional consequences using cellular assays

• Rescue experiments:

  • Reintroduce wild-type or mutant NFATC2IP using lentiviral vectors

  • Express tagged versions (3xFlag-tagged NFATC2IP) for detection

  • Validate expression using antibodies against both NFATC2IP and the tag

• Advanced applications:

  • Combine with ChIP-seq to map genome-wide binding sites

  • Use with proximity labeling techniques to identify novel interaction partners

What proteomics approaches can be combined with NFATC2IP antibodies for studying protein interactions?

For comprehensive analysis of NFATC2IP protein interactions:

• Co-immunoprecipitation strategies:

  • Use NFATC2IP antibodies for IP followed by mass spectrometry

  • Compare interactomes under different cellular conditions (resting vs. activated T cells)

  • Focus on interactions with NFAT family members and chromatin modifiers

• Proximity labeling approaches:

  • Generate BioID or TurboID fusions with NFATC2IP

  • Use antibodies to validate proximity labeling results

  • Compare proximity interactomes of wild-type vs. domain mutants

• Cross-linking mass spectrometry:

  • Apply protein cross-linking before immunoprecipitation

  • Identify direct binding interfaces between NFATC2IP and partners

  • Focus on UBC9/UBE2I interactions to understand SUMOylation regulation

• Validation of mass spectrometry results:

  • Confirm key interactions using reciprocal co-IP with NFATC2IP antibodies

  • Perform domain mapping to identify interaction regions

  • Use in vitro binding assays with recombinant proteins